3D bioprinting can create living human tissues on demand based on specifications contained in a digital file. Such highly customized, physiologically-relevant 3D human tissue models can screen potential drug candidates as an alternative to expensive pre-clinical animal testing. The Willerth lab has developed a novel fibrin-based bioink for bioprinting neural tissues derived from human induced pluripotent stem cells (hiPSCs), which can become any cell type found in the body. Our team uses Aspect Biosystem’s novel RX1 bioprinter featuring Lab-On-a-Printer™ (LOP) technology as it enables us to fabricate complex structures found in healthy neural tissues. The microfluidic LOP™ printhead cartridges generate cell-containing hydrogel fibers of defined diameters that are precisely deposited into defined 3D structures using a sheath fluid that triggers hydrogel cross-linking of the bioink. The sheath fluid also insulates cells within the fiber from shear stress, protecting fragile primary cells from shear-induced cell death. This process allows us to maintain high levels viability (>90% post printing) not previously seen in the literature. Here I will discuss the latest work from our group detailing the composition of our 3D bioprinted tissues and exciting avenues for future work.

The vast majority of microfluidic systems are built by replica-molding in elastomers (such as PDMS) or in thermoplastics (such as PMMA or polystyrene). However, biologists and clinicians typically do not have access to microfluidic technology because they do not have the engineering expertise or equipment required to fabricate and/or operate microfluidic devices. Furthermore, the present commercialization path for microfluidic devices is usually restricted to high-volume applications in order to recover the large investment needed to develop the plastic molding processes. Several groups, including ours, have been developing microfluidic devices through stereolithography (SL), a form of 3D printing, in order to make microfluidic technology readily available via the web to biomedical scientists. However, most available SL resins do not have all the favorable physicochemical properties of the above-named plastics (e.g., biocompatibility, transparency, elasticity, and gas permeability), so the performance of SL-printed devices is still inferior to that of equivalent PDMS devices. Inspired by the success of hydrogel PEG-DA biocompatibility, we have developed microfluidic devices by SL in advanced resins that share all the advantageous attributes of PDMS and thermoplastics so that we can 3D-print designs with comparable performance and biocompatibility to those that are presently molded.

A large percentage of drug candidates fail at the clinical trial stage due to a lack of efficacy and unacceptable toxicity, primarily because the in vitro cell culture models and in vivo animal models commonly used in preclinical studies provide limited information about how a drug will affect human physiology. The need for more physiologically relevant in vitro systems for preclinical efficacy and toxicity testing has led to a major effort to develop “Microphysiological Systems (MPS)”, aka tissue chips (TC), based on engineered human tissue constructs.

Microphysiological systems hold promise for improving therapeutic drug approval rates by providing more physiological, human-based, in vitro assays for preclinical drug development activities compared to traditional in vitro and animal models. The full impact of MPS technologies will be realized only when robust approaches for in vitro–in vivo (MPS-to-human) translation are developed and utilized, and explain how the burgeoning field of quantitative systems pharmacology (QSP) can fill that need.

Despite substantial investments to meet clinical and commercial expectations, and while scientific achievements at the preclinical research stage have sometimes been impressive, scaffold-based Tissue Engineering approaches are struggling to find the way to therapeutic and industrial success. Main challenges for the manufacturing of tissue engineered ATMPs concern the improvement of the standardisation of manufacturing processes, tissue functionality, and cost-effectiveness and profitability of related treatments.

Based on our experience in the field of bioprinting, we discuss how this technology – thanks to its characteristics resulting from the convergence of automation, biology and digital technology – should make it possible to overcome current tissue manufacturing bottlenecks and also provide new opportunities.

The development of methods for achieving spatiotemporal control over biomolecular gradients could enable advances in areas such as synthetic tissue engineering, biotic-abiotic interfaces, and bionanotechnology. Living organisms guide tissue development through orchestrated gradients of biomolecules that direct cell growth, migration, and differentiation. Our group has previously presented a method to 3D print stimuli-responsive core/shell capsules for programmable release of multiplexed gradients within hydrogel matrices. These capsules are comprised of an aqueous core, which can be formulated to maintain the activity of payload biomolecules, and a PLGA shell. The shell can be loaded with plasmonic gold nanorods (AuNRs), which permits selective rupturing of the capsule when irradiated with a laser wavelength determined by the lengths of the nanorods. This precise control over space, time, and selectivity allows for the ability to pattern 2D and 3D multiplexed arrays of content-loaded capsules, along with tunable laser-triggered rupture and release of payloads into a hydrogel ambient – allowing for dynamic tissue engineering applications. One particular example includes the use of these capsules in the development of 3D in vitro models capable of recapitulating native tumor microenvironments. Here, we build tumor constructs via the co-3D printing of living cells, natural hydrogels, and programmable release capsules. This enables the spatiotemporal control over signaling molecular gradients, thereby dynamically modulating cellular behaviors at the local level. Vascularized tumor models are created to mimic key steps of cancer dissemination (invasion, intravasation, and angiogenesis), based on guided migration of tumor cells and endothelial cells in the context of stromal cells and growth factors. These ‘4D printed’ vascularized tumor tissues provide a proof-of-concept dynamic tissue engineering platform to i) explore the molecular mechanisms of tumor progression and metastasis, and ii) preclinically identify therapeutic agents and screen anticancer drugs.

Corneal endothelial cells maintain visual acuity by regulating water content in the cornea. Damage to this region requires corneal transplant from donor tissue. Using 2-photon lithography for molding, a biomimetic membrane triggers pluripotent cells to differentiate into corneal endothelial like cells. This work with Fraunhofer IPT provides foundational understanding of mechanical cues required in this tissue. Our future directions involve scaling this work to translate production to clinical test levels.

Heart attack can lead to necrosis of myocardial tissue. Stem-cell therapy is an emerging strategy that involves the injection of cardiac progenitor cells to encourage tissue regrowth. However, ongoing trials have showed that the efficacy of this technique is limited and the retention rate of the delivered cells is poor. Cardiac tissue engineering includes the use of cells with biomaterials and signaling molecules to regenerate damaged heart tissue. Herein we present ongoing efforts to optimize cardiac and vascular tissue engineering approaches toward commercial and clinical applications. This includes utilization of additive manufacturing to systematize the process and create patient-matched therapies. These approaches have implications for the treatment of cardiovascular disease and precision medicine.

Current microfabrication techniques typically require complex, labor-intensive processes. An alternative method of economical and rapid prototyping is extrusion-based 3D printing. While 3D printing has more recently been applied to the field of microfluidics, channel resolution is poor. Furthermore, since the current generation of microfluidics devices require the integration of electronic components, we utilized dual extrusion-based 3D printing, thereby allowing for the prototyping of multi-material microfluidic devices with integrated electronics. Devices were designed in SolidWorks (modeling software), exported to BCN3D Cura (slicing program), and printed via BCN3D Sigma (dual-extrusion 3D printer). For devices with electronic components, conductive polylactic-acid (PLA) was inlaid within a non-conductive PLA framework to create an internal circuitry.

Functional open-faced microfluidics channels as small as 50 µm in width were produced. However, 100 µm width channels were more highly reproducible. Fully enclosed horizontal (200-500 µm) and vertical (750-1000 µm) channels were also fabricated. Hybrid devices contained both vertical and horizontal channels to create 3D fluidic arrays. Multi-layered electronic devices with multi-electrode microfluidic wells were created and allowed for simple electroanalysis. 3D printed electronic circuits were also used to thermally actuate paraffin valves in microfluidics channels, which may allow for the automation of multi-step reactions.

The talk will present the results obtained in a project that aims at encapsulating human and neo-natal pig islets, using chemically modified alginates with improved mechanical and biocompatibility properties, for type 1 diabetes cell therapy. The encapsulation is done in microfluidics, which gives better results of microcapsules sphericity, monodispersity and size.

Microfluidic Paper-Based Analytical Devices (MicroPADs) have emerged as useful diagnostic tools. They possess many key characteristics, including portability, cost-effectiveness, ease of use, low sample volume requirements, and ability to operate without supporting equipment. Researchers have modified microPADs in a myriad of ways to increase functionality, however, the use of 3D printing on microPADs has yet to be explored. The purpose of this project was to use 3D printing to expand microPAD functionality and combat current limitations. MicroPADs were fabricated on chromatography paper via wax printing and affixed to the bedplate of a BCN3D Sigma (dual-extrusion 3D printer). Polylactic acid (PLA) and conductive PLA structures were fabricated on microPADs via 3D printing. 3D fluid reservoirs were fabricated from PLA and allowed for larger sample volumes and further wicking distances on microPADs. Electrodes were fabricated from conductive PLA and allowed for simple electroanalysis on paper. Hybrid devices with both PLA channels (100 µm) and paper channels were also created. PLA-backed hemi- and fully-enclosed paper channels were fabricated, which may allow for increased robustness and reduced contamination of biochemical assays on microPADs, as well as the ability to run devices in direct contact with surfaces (i.e., not held in suspension).

We developed a polymer-and-glass chip for fast delivery of reagents on tissue slides and fully automatic imaging by integration with an optical microscope, along with high-throughput image-processing for single cell mapping. As proof-of-concept analyses, we identified co-expression and co-localization patterns of biomarkers to classify the immune cells and their activation status in lung adenocarcinoma.

As part of the European consortium BIOCAPAN, we present preliminary results of an innovative advance therapy medicinal product for the treatment of type 1 diabetes and some severe cases of type 2 diabetes based on microencapsulation of insulin secreting islets.

In this talk, I will present two different stories that illustrate the power of biomaterials in clinical applications. The first addresses a major challenge in developing surgical solutions for children with congenital heart disease. Current solutions involve multiple staged surgeries because the implants do not grow with the child. We have developed several methods to generate a layered tissue patch that mimics the cellular organization of native vessels and a bioMEMS device that can be used to assess physiological function of these tissue-engineered constructs with patient data input. The second story addresses our efforts to detect and prevent surgical adhesions, bands of tissue arising from abdominal surgeries that can lead to small bowel obstruction and infertility. The annual cost of adhesion-related complications to the US healthcare system is estimated to be as high as $5 billion. We have developed a novel formulation of ultrasound contrast agents that are stable for up to 4 days and target components found during the initial stage of adhesion formation.

18:30

Beer and Wine Reception and Networking

19:30

Close of Day 1 of the Conference

19:45

3D-Printing in Microfluidics Training Course Presented by Professor Albert Folch, University of Washington